One of the features of the immune system currently under study is the process by which the immune system "fails", and generates antibodies against so-called "self" antigens in the subject. This phenomenon leads to the development of the so-called autoimmune diseases which include arthritis, rheumatism, some forms of diabetes and the disease under discussion herein, scleroderma.
In the autoimmune diseases studied, one generally finds that the disease can be correlated to an immune response against a normal protein produced by the organism, a particular cell type, etc. The mechanism by which this takes place is under intensive investigation, but for now, most efforts center on the diagnosis of the conditions.
Diagnosis of an autoimmune disease most practically requires identification of antibodies against the particular protein. In theory, this is a very simple concept to understand. In practice it is difficult to provide appropriate diagnostic methodologies. Proteins produced by organisms such as humans are produced in extremely small quantities, and securing amounts of these proteins sufficient even for laboratory, research, involves large scale, difficult purification for vanishingly small yields. For example, purification of the naturally occurring hormone erythropoietin once required processing of 2500 liters of urine to secure a few milligrams of protein. Clearly, this is not an acceptable approach for diagnosis.
Given the developments in DNA technology, one approach to this problem has been to locate and to isolate nucleic acid sequences which code for or express the protein of interest. Again, in theory, the DNA, e.g., is isolated, transfected into host cells, and large quantities of protein produced. In practice, this is not always, and usually is not very simple. Among the issues confronting the investigator are: (i) the number of copies of the gene available, (ii) whether the gene is available in a cell type under consideration, (iii) assuming the gene is available, can it be identified, and (iv) can it be adapted to work in a non-source cell environment.
The issues around (i) and (ii) are self evident, and will not be discussed further. As to (iii), theory says that once one has even a small amount of protein available one secures its amino acid sequence and, using the genetic code, constructs oligonucleotide probes to locate the gene. Again, the practitioner knows that the issues around this theory include the problem of creating a probe that is precisely the right size to locate the desired gene and no other. This is not very easy to solve, and involves a great deal of trial and error.
If nucleic acids expressing or coding the protein of interest from another species are available, again, the theory says that, based on expected homology, one ought to be able to find, e.g., a human gene using the genes or fragments thereof from other species. When the protein of interest possesses a dissimilar sequence, however, this methodology will fail. Regarding (iv), supra, once a clone is isolated, there is no guarantee that it will work outside of its "home" environment.
With respect to the nucleic acids coding for fibrillarin, although proteins and DNA for other species were known, as per Lapeyre et al., Mol. Cell Biol. 10: 430-434 (1990) (Xenopus DNA); Henriquez et al., J. Biol. Chem. 265: 2209-2215 (1990) (yeast DNA), the disclosures of which are incorporated by reference herein, the normal protocols and theoretical considerations did not permit isolation of the human gene, nor the pure protein.
A methodology has been developed, however, which has permitted the inventor to identify and isolate nucleic acid sequences which code for and express human fibrillarin. In addition, it has been found that the isolated nucleic acid sequences will produce human fibrillarin in vitro, eliminating problems which may be encountered in in vivo systems. Finally, the protein itself has been produced and purified in quantities sufficient to permit characterization and use thereof.
These and other aspects of the invention are developed in detail via the disclosure which follows.